Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

• the present invention relates to compounds useful for inhibiting cyclin-dependent kinase 7 (CDK7), pharmaceutical compositions, and methods for treating diseases related to CDK7 activity.
• CDK7 cyclin-dependent kinase 7
• CDKs Cyclin-dependent kinases
• CDK7 binds to cyclin H and MATI to form a trimeric cyclin-activating kinase (CAK) that performs its function by phosphorylating other CDKs involved in cell -cycle control. These complexes control specific transitions between two subsequent phases in the cell cycle.
• CDK7 is implicated in both temporal control of the cell cycle and transcriptional activity.
• CDK7 is implicated in the transcriptional initiation process by phosphorylation of Rbpl subunit of RNA Polymerase II (RNAPII). Uncontrolled cell proliferation and deregulated transcription is a cancer hallmark.
• Targeting CDK7 selectively may offer an advantage by simultaneously inhibiting active transcription and cell-cycle progression. Therefore, CDK7 is a promising target for the treatment of cancer, in particular aggressive and hard- to-treat cancers.
• CDK7 inhibitors against CDK7 have been reported in the literature (see, e.g., WO 2015/154022, WO 2016/142855, WO 2016/160617, WO 2016/193939, and WO 2017/044858). There remains a need to provide CDK7 inhibitors which can be used in the treatment of cell proliferative disorders, such as cancer. Additionally, there is a need to provide CDK7 inhibitors which are selective for CDK7 compared to other CDKs.
• the present invention provides novel compounds that are selective CDK7 inhibitors. Such new compounds could address the need for potent, effective treatment of cancer, especially cancer with deregulated transcription.
• the present invention could also address the need for potent, effective treatment of urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, and/or gliomas.
• the present invention provides a compound of formula:
• a pharmaceutically acceptable salt thereof Especially preferred is a besylate salt. Also preferred is the hemi-edisylate hydrate salt.
• the present invention also provides a method for the treatment of cancer, in particular for the treatment of cancer with deregulated transcription.
• the cancer is urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas.
• the cancer is colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer.
• the cancer is breast cancer.
• the present invention also provides a method of treating urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas in a patient, comprising testing for the presence of at least one loss of function mutation in the ARID 1 A, KMT2C, KMT2D and/or RBI genes in a biological sample from the patient and administering a therapeutically effective amount of a compound or salt of the present invention, in particular [(3S)-l-[(E)-4-(dimethylamino)but-2-enoyl]pyrrolidin- 3-yl] 4-[(3-isopropyl-5-methyl-pyra
• the salt is a besylate salt or a hemi-edisylate hydrate salt.
• the cancer is colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer. Most preferably, the cancer is breast cancer.
• the biological sample is a tumor sample and the sample is assayed by genomic/DNA sequencing.
• the sample is obtained from the patient prior to a first administration of the compound or salt thereof, preferably [(3S)-l-[(E)-4- (dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carboxylate or a pharmaceutically acceptable salt thereof, to the patient.
• the salt is a besylate salt or a hemi-edisylate hydrate salt.
• the gene is the ARID 1 A gene.
• the gene is the KMT2C gene.
• the gene is the KMT2D gene.
• the gene is the RBI gene.
• the present invention also provides a method of treating urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas in a patient, comprising administering a therapeutically effective amount of a compound or salt of the present invention, in particular [(3S)-l-[(E)-4-(dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5- methyl-pyrazolo[l,5-a]pyrimidin-7-yl)amino]piperidine-l-carboxylate or a
• the salt is a besylate salt or a hemi-edisylate hydrate salt.
• the cancer is colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer. Most preferably, the cancer is breast cancer.
• the biological sample is a tumor sample and the sample is assayed by genomic/DNA sequencing.
• the sample is obtained from the patient prior to a first administration of the compound or salt thereof, preferably [(3S)-l-[(E)-4- (dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carboxylate or a pharmaceutically acceptable salt thereof, to the patient.
• the salt is a besylate salt or a hemi-edisylate hydrate salt.
• the gene is the ARID 1 A gene.
• the gene is the KMT2C gene.
• the gene is the KMT2D gene.
• the gene is the RBI gene.
• the present invention also provides a method of treating urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas in a patient, comprising administering a therapeutically effective amount of a compound or salt of the present invention, in particular [(3S)-l-[(E)-4-(dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5- methyl-pyrazolo[l,5-a]pyrimidin-7-yl)amino]piperidine-l-carboxylate
• the salt is a besylate salt or a hemi-edisylate hydrate salt.
• the cancer is selected from the group consisting of colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer. Most preferably, the cancer is breast cancer.
• the biological sample is a tumor sample and the sample is assayed by genomic/DNA sequencing.
• the sample is obtained from the patient prior to a first administration of the compound or salt thereof, preferably [(3S)-l-[(E)-4- (dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carboxylate or a pharmaceutically acceptable salt thereof, to the patient.
• the salt is a besylate salt or a hemi-edisylate hydrate salt.
• the gene is the ARID 1 A gene.
• the gene is the KMT2C gene.
• the gene is the KMT2D gene.
• the gene is the RBI gene.
• the present invention also provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.
• the composition further comprises one or more other therapeutic agents.
• the present invention provides a pharmaceutical composition for the treatment of cancer comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with one or more
• the present invention provides a pharmaceutical composition for the treatment of cancer with deregulated transcription comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.
• the cancer is urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas.
• the cancer is colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer. In a more preferred embodiment, the cancer is breast cancer.
• the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer also comprising performing an in vitro assay using a biological sample from the patient, determining the presence of at least one inactivating mutation in the ARID 1 A, KMT2C, KMT2D and RBI genes, and administering a therapeutically effective amount of the compound or salt thereof to the patient if at least one inactivating mutation in any of the genes is present.
• the cancer is fo ruse in the treatment of urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas.
• the biological sample is a tumor sample and the sample is assayed by genomic/DNA sequencing.
• the compound of salt thereof is administered to the patient at a dose of about 1 mg to 2 g.
• the sample is obtained from the patient prior to the first administration of the compound or the salt thereof to the patient.
• a patient is selected fro havin an inactivating mutation in the ARID 1 A gene.
• the patient is selected for having an inactivating mutation in the KMT2C gene.
• a patient is selected for having an inactivating mutation in the KMT2D gene.
• a patient is selected for having an inactivating mutation in the RBI gene.
• the present invention provides a compound of the invention, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for the treatment of cancer with deregulated transcription.
• the present invention provides the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer with deregulated transcription.
• the cancer is urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas.
• the cancer is selected colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer.
• the cancer is breast cancer.
• the present invention provides a compound of the invention, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for the treatment of cancer.
• the present invention provides the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer.
• the cancer is urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas.
• the cancer is colorectal cancer, breast cancer, lung cancer, ovarian cancer, or gastric cancer.
• the cancer is breast cancer.
• the present invention provides for the manufacture of a medicament for the treatment of a urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas, also comprising performing an in vitro assay using a biological sample from the patient, determining the presence of at least one inactivating mutation in the ARID 1 A, KMT2C, KMT2D and RBI genes, and administering a therapeutically effective amount of the compound or salt thereof to the patient if at least one inactivating mutation in any of the genes is present.
• the biological sample is a tumor sample and the sample is assayed by genomic/DNA sequencing.
• the compound of salt thereof is administered to the patient at a dose of about 1 mg to 2 g.
• the sample is obtained from the patient prior to the first administration of the compound or the salt thereof to the patient.
• a patient is selected for having at least one inactivating mutation in the ARID 1 A gene.
• a patient is selected for having at least one inactivating mutation in the KMT2C gene.
• a patient is selected for having at least one inactivating mutation in the KMT2D gene.
• a patient is selected for having at least one inactivating mutation in the RBI gene.
• the present invention provides a compound of the invention [(3S)-l-[(E)-4- (dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carboxylate in a crystalline salt form.
• the present invention also provides crystalline [(3S)-l-[(E)-4-(dimethylamino)but-2-enoyl]pyrrolidin- 3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5-a]pyrimidin-7-yl)amino]piperidine-l- carboxylate hemi-edisylate hydrate.
• the present invention also provides [(3S)-l-[(E)-4- (dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carboxylate hemi-edisylate hydrate in a crystalline form characterized by a X-ray powder diffraction pattern having characteristic peaks using CuKa radiation, in 2Q ⁇ 0.2°, occurring at 18.5° in combination with one or more peaks selected from the group consisting of 21.5°, 16.7°, and 15.2°.
• the present invention also provides crystalline [(3S)-l-[(E)-4-(dimethylamino)but-2-enoyl]pyrrolidin- 3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5-a]pyrimidin-7-yl)amino]piperidine-l- carboxylate besylate.
• the present invention also provides [(3S)-l-[(E)-4- (dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carboxylate besylate in a crystalline form
• the present invention also provides crystalline [(3S)-l-[(E)-4-(dimethylamino)but-2-enoyl]pyrrolidin-3-yl] 4-[(3- isopropyl-5-methyl-pyrazolo[l,5-a]pyrimidin-7-yl)amino]piperidine-l-carboxylate hydrochloride.
• the present invention also provides [(3S)-l-[(E)-4-(dimethylamino)but-2- enoyl]pyrrolidin-3-yl] 4-[(3-isopropyl-5-methyl-pyrazolo[l,5-a]pyrimidin-7- yl)amino]piperidine-l-carboxylate hydrochloride in a crystalline form characterized by a X-ray powder diffraction pattern having characteristic peaks using CuKa radiation, in 2Q ⁇ 0.2°, occurring at 18.9° in combination with one or more peaks selected from the group consisting of 5.5°, 15.5°, and 9.7°.
• the present invention also encompasses intermediates and processes useful for the synthesis of a compound of the present invention.
• treating refers to restraining, slowing, stopping, or reversing the progression or severity of an existing symptom, condition or disorder.
• cancer and“cancerous” refer to or describe the physiological condition in patients that is typically characterized by unregulated cell proliferation. Included in this definition are benign and malignant cancers.
• By“early stage cancer” or“early stage tumor” is meant a cancer that is not advanced or metastatic or is classified as a Stage 0, 1, or II cancer.
• cancer examples include, but are not limited to, urothelial cancer, uterine cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, cervical cancers, prostate cancer, haemotological cancers, sarcomas, skin cancers, or gliomas.
• a compound of the present invention may react to form pharmaceutically acceptable salts.
• Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art (see, e.g., P. Stahl, el al. Handbook of
• the skilled artisan will also appreciate that the Cahn-Ingold-Prelog (R) or (S) designations for all chiral centers will vary depending upon the substitution patterns of the particular compound.
• the single enantiomers may be prepared beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively, the single enantiomers may be isolated from mixtures by standard chiral chromatographic or crystallization techniques at any convenient point in the synthesis of compounds of the invention. Single enantiomers of compounds of the invention are a preferred embodiment of the invention.
• a compound of the present invention is preferably formulated as pharmaceutical compositions administered by a variety of routes.
• Such pharmaceutical compositions and processes for preparing the same are well known in the art (see, e.g. , Remington: The Science and Practice of Pharmacy (A. Gennaro, et al, eds., 21 st ed., Mack Publishing Co., 2005)). More particularly preferred, is a pharmaceutical composition comprising a compound of the formula,
• An especially preferred embodiment of the present invention relates to the compound, (3S)-l-[(2E)-4-(Dimethylamino)but-2-enoyl]pyrrolidin-3-yl 4- ⁇ [5-methyl -3- (propan-2-yl)pyrazolo[l,5-a]pyrimidin-7-yl]amino ⁇ piperidine-l-carboxylate:
• Another especially preferred embodiment of the present invention relates to the compound, (3S)-l-[(2E)-4-(Dimethylamino)but-2-enoyl]pyrrolidin-3-yl 4- ⁇ [5-methyl-3- (propan-2-yl)pyrazolo[l,5-A]pyrimidin-7-yl]amino ⁇ piperidine-l-carboxylate:
• a further especially preferred embodiment of the present invention relates to the compound, (3R)-l-[(2E)-4-(Dimethylamino)but-2-enoyl]pyrrolidin-3-yl 4- ⁇ [5-methyl-3- (propan-2-yl)pyrazolo[l,5-A]pyrimidin-7-yl]amino ⁇ piperidine-l-carboxylate (as shown by (III) below):
• Another especially preferred embodiment of the present invention relates to the compound, (3R)-l-[(2E)-4-(Dimethylamino)but-2-enoyl]pyrrolidin-3-yl 4- ⁇ [5-methyl-3- (propan-2-yl)pyrazolo[l,5-A]pyrimidin-7-yl]amino ⁇ piperidine-l-carboxylate:
• the compounds of the present invention are generally effective over a wide dosage range.
• dosages per day fall within the range of about 1 mg to about 2 g.
• dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed while maintaining a favorable benefit/risk profile, and therefore the above dosage range is not intended to limit the scope of the invention in any way.
• the amount of the compound actually administered will be determined by a physician, in light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
• variable protecting group may be the same or different in each occurrence depending on the particular reaction conditions and the particular
• Ethylenediaminetetraacetic acid “ATCC” refers to American Type Culture Collection; “RT” refers to room temperature;“PBS” refers to phosphate-buffered saline;“BSA” refers to bovine serum albumin;“FBS refers to fetal bovine serum;“RNAase” refers to ribonuclease;“cDNA” refers to complementary DNA;“GST” refers to glutathione S- transferase;“His” refers to histidine;“GSH” refers to glutathione; and“HBSS” refers to Hank’s Balanced Salt Solution.
• the compounds of the invention, or pharmaceutically acceptable salts thereof may be prepared by a variety of procedures known in the art, as well as the Preparations and Examples below.
• the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the invention, or pharmaceutically acceptable salts thereof.
• the products of each step in the schemes below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization.
• the reagents and starting materials are readily available to one of ordinary skill in the art.
• the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology and habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. See, e.g. The United States Pharmacopeia #23, National Formulary #18, pages 1843-1844, 1995.
• the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature or humidity at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard.
• a peak position variability of ⁇ 0.2 in 20 will take into account these potential variations without hindering the unequivocal identification of the indicated crystal form.
• Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in units of ° 20), typically the more prominent peaks. Adjust the crystal form diffraction patterns, collected at room temperature and relative humidity, based on NIST 675 standard peaks at 8.853 and 26.774 degrees 2-theta.
• a prepared sample of the crystalline hemi-edisylate hydrate is characterized by an XRD pattern using CuKa radiation as having diffraction peaks (2 -theta values) as described in the table below, and in particular having a peak at 18.5° in combination with one or more peaks selected from the group consisting of 21.5°, 16.7°, and 15.2°; with a tolerance for the diffraction angles of 0.2 degrees.
• a prepared sample of the crystalline besylate is characterized by an XRD pattern using CuKa radiation as having diffraction peaks (2 -theta values) as described in the table below, and in particular having a peak at 21.5° in combination with one or more peaks selected from the group consisting of 12.4°, 17.3°, and 15.8°; with a tolerance for the diffraction angles of 0.2 degrees.
• a prepared sample of the crystalline hydrochloride hydrate is characterized by an XRD pattern using CuKa radiation as having diffraction peaks (2 -theta values) as described in the table below, and in particular having a peak at 18.9° in combination with one or more peaks selected from the group consisting of 5.5°, 15.5°, and 9.7°; with a tolerance for the diffraction angles of 0.2 degrees.
• the following assays demonstrate that a compound of the invention is an inhibitor of CDK7 activity.
• the results of the assays also show that a compound of the invention inhibits CDK7 signaling in the cancer cells. Additionally, a compound of the invention inhibits proliferation in cancer cell lines and tumor growth in xenograft tumor model of cancer.
• IC 50 refers to the concentration of an agent that produces 50% of the maximal inhibitory response possible for that agent or, alternatively, to the concentration of an agent which produces 50% displacement of ligand specific binding to the receptor;
• Relative IC 50 values are determined using fluorescence unit by calculating percent inhibition with respect to on-plate“MIN” and“MAX” controls and then fitting the ten- point dose response data to a four-parameter logistic equation.
• the purpose of this assay is to measure the ability of a compound of the invention to inhibit CDK7/CyclinH/Matl complex kinase activity.
• the biochemical assays are performed with no preincubation of the enzyme with the compound or with 3 hours preincubation.
• Functional assays provide support on whether the compounds of the present invention exhibit the ability to inhibit the CDK7 and CDK9 kinase activities. All ligands, solvents, and reagents employed in the following assays are readily available from commercial sources, or can be readily synthesized by one skilled in the art.
• the IC 50 determination for CDK7 and CDK9 are determined as follows.
• the IC 50 activity of the inhibitor is determined using radiolabel filter binding (FB) assays using the purified human recombinant enzyme in the presence of ATP//[ 33 P] ATP and peptide substrate.
• FB radiolabel filter binding
• Reactions are carried out in 96 well polystyrene plates in a final volume of 25 pL per well. 5 pL of test compound in 20% DMSO, 10 pL of substrate solution (ATP/33PATP and CDK7/9 tide) and 10 pL of enzyme solution are mixed. The substrate solution is prepared to give a final concentration of 100 mM ATP/[ 33 P]ATP (NEN).
• Test compounds are serially diluted 1 :3 in 20% DMSO to create a 10 point curve at a starting concentration of 20 mM.
• 20% DMSO buffer alone without test compound is employed as high control (full activity in the absence of any inhibitor), 500 mM EDTA is used to determine the level of background in the absence of enzyme activity (low control).
• 500 mM EDTA is used to determine the level of background in the absence of enzyme activity (low control).
• After mixing 5 pL of compounds with 10 pL of enzyme solution the plate is incubated for 0 or 180 minutes at 22 °C. After that time the reaction is initiated by the addition of 10 pL substrate solution and incubated for 50 minutes at 22 °C.
• the reaction is terminated by the addition of 80 pL of cold 10% ortophosphoric solution.
• the Filter Plates (opaque, non-sterile filter plates) are prewashed with 10 pL of 10% orthophosphoric solution to each well. 100 pL of the mixture are transferred to a phosphocellulose filter and incubated at room temperature for 45 minutes. Filter plates are washed with 200 pL 0.5 % orthophosphoric acid 3 times on a filter plate processor. Incorporation of 33Pi (counting of“cpm”) is determined by adding 80 pL of MICROSCINTTM to each well and read on a counter after an hour. Data is processed through a GENED ATA SCREENER ® tool.
• Y hot + [(top-bot)/l+(x/ IC 5 o)slope]
• Y % inhibition
• X concentration yielding y% inhibition
• Bottom minimum value of y attained by curve
• Top maximum value of y attained by curve
• Slope steepness of curve at IC 50.
• IC 50 concentration of compound that reduces a given response (ligand binding, enzyme response) by 50%.
• Examples 1 and 3 display an IC 50 of 0.0173 mM and 0.0487 pM in CDK7 without preincubation, respectively. After 3 hours of preincubation of CDK7 enzyme with Examples 1 and 3, they show an IC 50 of 0.00237 mM and 0.00506 pM, respectively. These data show that both Examples 1 and 3 inhibit CDK7.
• Assay for inhibition of CDK9/CyclinTl kinase activity Assay for inhibition of CDK9/CyclinTl kinase activity:
• the IC 50 activity of the inhibitor is determined using radiolabel filter binding (FB) assays using the purified human recombinant enzyme in the presence of ATP and peptide substrate.
• FB radiolabel filter binding
• Reactions are carried out in 96 well polystyrene plates in a final volume of 25 pL per well. 5 pL of test compound in 20% DMSO, 10 pL of substrate solution (ATP//[ 33 P]ATP and CDK7/9 tide) and 10 pL of enzyme solution are mixed.
• the substrate solution is prepared to give a final concentration of 100 pM ATP/[ 33 P]ATP (NEN lOuCi/pL, 3000 Ci/mmol) and 200 pM CDK7/9 peptide ((YSPTSPSYSPTSPSYSPTSPSKKKK) (SEQ ID NO: 1)) diluted in kinase buffer of 4 mM MgCh, 0.0025% TRITONTM X-100, 1.58 mM DTT and 15.80 mM HEPES.
• the enzyme solution is prepared for a final concentration of 7.5 nM CDK9/cyclinTl enzyme [Proqinase 0371-0345-1 (Lot 004)] diluted in kinase buffer.
• Test compounds are serially diluted 1 :3 in 20% DMSO to create a 10 point curve at a starting concentration of 20 pM.
• 20% DMSO buffer alone without test compound is employed as high control (full activity in the absence of any inhibitor), 500 mM EDTA is used to determine the level of background in the absence of enzyme activity (low control).
• 500 mM EDTA is used to determine the level of background in the absence of enzyme activity (low control).
• After mixing 5 pL of compounds with 10 pL of enzyme solution the plate is incubated for 0 or 180 minutes at 22 °C. After that time the reaction is initiated by the addition of 10 pL substrate solution and incubated for 60 minutes at 22 °C. The reaction is terminated by the addition of 80 pL of cold 10% ortophosphoric solution.
• Filter plates (opaque, non-sterile filter plates) are prewashed with 10 pL of 10% orthophosphoric solution per well. 100 pL of the mixture are transferred to a phosphocellulose filter and incubate at room temperature for 45 minutes. Filter plates are washed with 200 pL 0.5 % orthophosphoric acid 3 times on a filter plate processor. 80 pL of MICROSCINTTM is added to each well and read on a scintillation counter after an hour. Data is processed through a GENEDATA-SCREENER ® tool.
• Y hot + [(top-bot)/l+(x/ ICso)slope]
• Y % inhibition
• X concentration yielding y% inhibition
• Bottom minimum value of y attained by curve
• Top maximum value of y attained by curve
• Slope steepness of curve at IC 50.
• %Inh [(median Max- xJ median Max - median Min)] ⁇ 100
• IC 50 concentration of compound that reduces a given response (ligand binding, enzyme response) by 50%.
• IC 50 relative concentration giving half the compound’s maximum response.
• Examples 1 and 3 display an IC 50 of 5.93 mM and 2.45 mM for CDK9 (3 hours preincubation), respectively. These data show that Examples 1 and 3 do not potently inhibit CDK9 activity.
• the purpose of these assays is to measure the ability of compounds to inhibit CDK7 and CDK9 signaling in cancer cells in vitro.
• HCT116 cells (ATCC CCL-247) are cultured in McCoy’s 5A Medium Modified media supplemented with 10% FBS, 1% NaPyr and 1% Pen/Strep and plated (prior to becoming 70% confluent) in 96-well flat-bottom plates at a density of 5,000 cells per well in 100 pL volume. The cells are then incubated overnight in a cell culture incubator (5% CO 2 , 95% Relative Humidity (RH) and 37 °C) and allowed to attach to the plate. The following morning the cells are dosed with compounds. Compound inhibitors are first solubilized at 60 pM in culture medium containing 0.6% DMSO.
• compound serial dilutions (1 :3) are prepared over a 60 mM to 0.003 mM range.
• Cells are dosed with the addition of 50 pL from serial dilution plate to assay plate containing cells attached with 100 pL of media producing a final DMSO concentration of 0.2% with a final compound concentration dose range between 20 and 0.001 pM.
• a reference compound diluted at 0.83 pM final concentration in the growth media containing 0.2% DMSO is used for max point media containing 0.2% of DMSO is used.
• the cell plates are incubated at 37 °C and 5% CO 2 for 4 hours.
• the growth media is removed carefully and the cells are fixed by adding 100 pL of 4% para-formaldehyde for 30 minutes at RT.
• Cells are washed once with PBS and incubated with 100 pL of cold MeOH for 15 minutes at RT for cell permeation.
• Cells are washed twice with PBS (100 pL/each) and blocked with 100 pL/well of 1% BSA/PBS for 30 minutes at RT.
• Fluorescence plates are scanned with ACUMEN EXPLORERTM [Laser scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD] to measure anti-phospho-carboxyl terminal domain at Serine 2 (pCTD).
• Image analysis is based on cellular fluorescent signals for identifying positive cells.
• pCTD (S2) positive cells are identified by mean intensity at 500-530 above the threshold. Total intensity at 575-640 from propidium iodide/DNA is used to identify individual cells.
• Assay output is % pCTD positive cells.
• the IC 50 is determined by curve fitting to a four parameter logistic for each output using GENE DATATM.
• the compounds described in Examples 1 and 3 display a relative IC 50 >20 mM and 3.52 pM for phosphoCTD (S2), respectively. These data show that both Examples 1 and 3 do not potently inhibit CDK9 in the cells.
• HCT116 cells (ATCC CCL-247) are cultured in McCoy’s 5A Medium Modified media supplemented with 10% FBS, 1% NaPyr and 1% Pen/Strep and plated (prior to becoming 70% confluent) in 96-well flat-bottom plates at a density of 5,000 cells per well in 100 pL volume. The cells are incubated overnight in a cell culture incubator (5% CO 2 , 95% Relative Humidity (RH) and 37 °C) and allowed to attach to the plate. The following morning, the cells are dosed with compounds. Compound inhibitors are solubilized at 60 mM in culture medium containing 0.6% DMSO.
• compound serial dilutions (1 :3) are prepared over a 60 mM to 0.003 pM range.
• Cells are dosed with the addition of 50 pL from serial dilution plate to assay plate containing cell attached with 100 pL of media producing a final DMSO concentration of 0.2% with a final compound concentration dose range between 20 and 0.001 pM.
• a reference compound diluted at 0.83 pM final concentration in the growth media containing 0.2% DMSO is used for max point media containing 0.2% of DMSO is used.
• the cell plates are incubated at 37 °C and 5% C0 2 for 4 hours. Growth media is removed carefully and the cells are fixed by adding 100 pL of 4% para-formaldehyde for 30 minutes at RT. Cell are washed once with PBS and incubated with 100 pL of cold MeOH for 15 minutes at RT for cell permeation. Again cells are washed twice with PBS (100 pL/each) and blocked with 100 pL/well of 1% BSA/PBS for 30 min at RT.
• Fluorescence plates are scanned with ACUMEN EXPLORERTM [Laser-scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD] to measure anti-phospho-carboxyl terminal domain at Serine 5 (pCTD). Image analysis is based on cellular fluorescent signals for identifying positive cells.
• pCTD (S5) positive cells are identified by mean intensity at 500-530 above the threshold. Total intensity at 575-640 from propidium iodide/DNA is used to identify individual cells. Assay output is % pCTD positive cells. The IC 50 is determined by curve fitting to a four parameter logistic for each output using GENE DATATM.
• HCT116 cells (ATCC CCL-247) are cultured in McCoy’s 5A Medium Modified media supplemented with 10% FBS, 1% NaPyr and 1% Pen/Strep and plated (prior to becoming 70% confluent) in 96-well flat-bottom plates at a density of 5,000 cells per well in 100 pL volume. The cells are then incubated overnight in a cell culture incubator (5% C0 2 , 95% Relative Humidity (RH) and 37 °C) and allowed to attach to the plate. The following morning the cells are dosed with compounds. Compound inhibitors are solubilized at 60 pM in culture medium containing 0.6% DMSO.
• compound serial dilutions (1 :3) are prepared over a 60 pM to 0.003 pM range.
• Cells are dosed with the addition of 50 pL from serial dilution plate to assay plate containing cell attached with 100 pL of media producing a final DMSO concentration of 0.2% with a final compound concentration dose range between 20 pM and 0.001 pM.
• a reference compound diluted at 0.83pM final concentration in the growth media containing 0.2% DMSO is used.
• the cell plates are incubated at 37 °C and 5% CO 2 for 4 hours.
• ACUMEN EXPLORERTM Laser-scanning fluorescence microplate cytometer manufactured by TTP LABTECH LTD] to measure anti-phospho-carboxyl terminal domain at Serine 5 (pCTD).
• Image analysis is based on cellular fluorescent signals for identifying positive cells.
• pCTD (S5) positive cells are identified by mean intensity at 500-530 above the threshold.
• Total intensity at 575-640 from propidium iodide/DNA is used to identify individual cells.
• Assay output is % pCTD positive cells.
• the IC 50 is determined by curve fitting to a four parameter logistic for each output using GENE DATATM.
• Examples 1 and 3 display a Relative IC 50 of 0.0828 mM and 0.0573 mM for cMyc. These data show that both Examples 1 and 3 inhibit the transcription of cMyc in HCT116 cells.
• Kinase inhibition profile of compound is determined by measuring residual activity values at four concentrations in singlicate in 320 wild-type protein kinase assays. The compounds are tested at 20 mM, 2 pM, 0.2 pM and 0.02 pM in singlicate. The final DMSO concentration in all reaction cocktails (including high and low controls) is 1%.
• a radiometric protein kinase assay (33PANQINASE ® Activity Assay, ProQinase) is used for measuring the kinase activity of the 320 protein kinases. All kinase assays are performed in 96-well FLASHPLATESTM in a 50 pL reaction volume. The reaction cocktail is pipetted in 4 steps in the following order:
• the assay for all protein kinases contain 70 mM HEPES-NaOH pH 7.5, 3 mM MgCh, 3 mM MnCh, 3 pM Na-orthovanadate, 1.2 mM DTT, ATP (variable amounts, corresponding to the apparent ATP -Km of the respective kinase, see Table 1), [g-33R]- ATP (approx. 8 x 1005 cpm per well), protein kinase (variable amounts; see Table 1), and substrate (variable amounts; see Table 1). All PKC assays (except the PKC-mu and the PKC-nu assay) additionally contain 1 mM CaCh,4 mM EDTA, 5 pg/mL
• the CAMK1D, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CAMK4, CAMKK1, CAMKK2, DAPK2,EEF2K, MYLK, MYLK2 and MYLK3 assays additionally contain 1 pg/mL Calmodulin and 0.5 mM CaCh.
• the PRKG1 and PRKG2 assays additionally contain 1 mM cGMP.
• the DNA- PK assay additionally contained 2.5 pg/mL DNA.
• the protein kinase reaction cocktails are incubated at 30 °C for 60 minutes. The reaction is stopped with 50 pL of 2% (v/v) H 3 PO 4 , plates are aspirated and washed two times with 200 pL 0.9% (w/v) NaCl. Incorporation of 33Pi (counting of“cpm”) is determined with a microplate scintillation counter. All protein kinase assays are performed with a BeckmanCoulter BIOMEK ® 2000/SL robotic system. All protein kinases provided by ProQinase are expressed in Sf9 insect cells or in E.coli as
• kinases are produced from human cDNAs and purified by either GSH-affmity chromatography or immobilized metal. Affinity tags are removed from a number of kinases during purification. The purity of the protein kinases is examined by SDS-PAGE/Coomassie staining, the identity is checked by mass spectroscopy.
• Non Standard IC 50S are calculated using a customized excel spreadsheet in conjunction with XLFit Add-in. Due to the low number of data points, (4) XL-Fit calculates a non standard IC 50 using a four parametric equation where three parameters are locked to fixed values.
• X The independent variable (i.e. what you control, such as, dose, concentration, etc.)
• the way to calculate the Non Standard IC 50 is to assign random values to C parameter and repeat it iteratively.
• the algorithm measures the differences in the sum of the residuals squared and will look for successive consecutive iterations where the change in the residuals is converging. Once the convergence limit has been met, the solution is regarded as the optimum and the fitting process ends.
• CDK12 and CDK13 are tested essentially as above but separately at 10 concentrations (2 x 10 5 M to 6 x 10 10 M) using semi-logarithmic dilutions.
• concentrations 2 x 10 5 M to 6 x 10 10 M
• concentrations 2 x 10 5 M to 6 x 10 10 M
• IC 50 values are calculated using QUATTRO ® WORKFLOWTM V3.1.1.
• the fitting model for the IC 50 determinations was "Sigmoidal response (variable slope)" with parameters "top” fixed at 100% and “bottom” at 0%.
• the fitting method used was a least-squares fit. Data are shown in Table 1 below.
• the data in Table 2 shows that the compound of Example 1 inhibits proliferation and viability of the specified tumor cells lines.
• Cell lines are plated at the density 5000 cells per well in 100 pL per well growth medium into a white 96-well cell culture plate. See Table 2 for cell line and culture medium information. Plates are incubated at 37 °C and 5% C0 2.
• a serial dilution of the test compound is prepared by diluting the compound 1 :3 in DMSO for 10 points.
• the DMSO plate is 1000X the final concentration.
• a DMSO alone column is included as a maximum growth control and 10 mM staurosporine final column is included as a maximum growth inhibition control.
• a 10X dilution plate is then prepared by adding 2 pL per well from the 1000X DMSO plate to 198 pL per well of OMEM (Life
• Cells are treated with indicated compound by adding 11 pL per well from the 10X OMEM plate to the cell plate containing 100 pL per well growth medium for a IX final concentration. Plates are placed back into the incubator at 37 °C and 5% C0 2. Seven days after compound addition, plates are removed from the incubator and allowed to equilibrate to RT. CELL TITER GLO ® reagent is thawed at room temperature and then prepare by mixing one vial of assay buffer with one vial of substrate and swirl gently to mix.
• CELL TITER GLO ® reagent is then added to the cell plate, lOOpL per well, and place on a Titer Plate Shaker at speed setting 2 for 15 minutes at room temperature. After 15 minute incubation on shaker, luminescence is read, 1 second per well, using a Wallac VICTOR2TM. Nonlinear regression and sigmoidal dose-response curves are used to calculate the half maximal inhibitory concentration (IC 50 ) with Graphpad Prism 6 software. Table 2
• Example II inhibits the in vitro growth of cancer cell lines from a variety of histologies including colon, breast, lung, ovary and stomach, in a dose dependent manner.
• This assay is to measure reduction in tumor volume in response to the compound of Example 1.
• multiple xenograft tumor models are utilized. Briefly, 5-10 x 10 6 tumor cells in a 1 : 1
• MATRIGEL® mix (0.2 mL total volume) are injected subcutaneously into the female athymic nude mice (Envigo, Harlan laboratories) for a majority of xenograft tumor models. Alternate mice strains are utilized to establish MDAMB468 (NOD SCID Gamma, Jackson labs), CT26 and EMT6 (B ALB/c, Envigo, Harlan Laboratories) xenografts. After allowing tumors to reach a desired size of ⁇ 300-500 mm 3 , animals are randomized into groups of 6-8 for efficacy studies. Test compound is administered via oral gavage (PO) at indicated doses and regimens. Tumor growth and body weight are monitored over time to evaluate efficacy and signs of toxicity.
• PO oral gavage
• Test compound is formulated in 5% N-methyl-2-pyrrolidone (NMP) in 1% hydroxyethylcellulose, 0.25% polysorbate 80, 0.05% antifoam in purified water (HEC) and administered by oral gavage (final volume 0.2 mL) at the doses indicated in Table 4.
• NMP N-methyl-2-pyrrolidone
• HEC purified water
• a test compound is formulated on a weekly basis and stored at 4 °C. Vehicles are administered to the control groups according the schedules used above using a volume of 0.2 mL per dose. Mice are dosed via oral gavage and tumor samples are collected at termination and stored at -80 °C.
• DBS dried blood spot
• Tumors are collected at study termination, cut into 3 sections and either snap frozen for exposure and protein analysis, or placed in RNAlater® for RNA analysis. Tissue samples are frozen and stored at -80 °C.
• Example 1 demonstrates significant anti -tumor activity in human cancer xenograft models (Table 3).
• Table 3 Summary of Example 1 in-vivo single-agent efficacy (DT/C) across variety
• Delta T/C% is calculated when the endpoint tumor volume in a treated group is at or above baseline tumor volume.
• the formula is l00*(T-T0)/(C-C0).
• T and C are mean endpoint tumor volumes in the treated or control group, respectively.
• TO and CO are mean baseline tumor volumes in those groups. *: Significant (p ⁇ 0.05)
• Cancer cell lines are profiled to evaluate the anti-proliferative activity of a test compound in-vitro. Mutation, copy number and gene expression information of ARID 1 A, KMT2C, KMT2D and/or RBI genes across cancer cell lines is obtained from COSMIC database (cancer.sanger.ac.uk) and cBioportal (http://www.cbioportal.org/). Cells are cultured in growth medium and plated into a 96-well plate in 100 pL/well growth medium at 5000 cells/well then incubated at 37 °C, 5% C0 2 overnight.
• the 1000X intermediate dilution plate is prepared by making a 10 mM working solution of a test sample in DMSO and performing 1 :3 dilutions in DMSO for 10 points.
• the 10X dosing plate is prepared by adding 2 pL from the 1000X intermediate dilution plate to 198 pL of OPTIMEM® + 10% FBS and mixing well.
• the cell plate is then treated by adding 11 pL from the 10X dosing plate into the 100 pL/well cell plate for a IX final concentration. Staurosporine is used as a maximum growth inhibition control at a final concentration of 5 pM.
• the cell plate is incubated for 7 days at 37 °C, 5% C0 2. Seven days after treatment, Cell Titer- Glo® (Promega cat# G7571) assay buffer and substrate are removed from -20 °C and allowed to equilibrate to RT. Assay buffer is added to the substrate and swirled gently to mix. The CELL TITER-GLO® reagent (100 pL/well) is added to the cell plate and incubated at RT for 15 minutes. After 15 minutes, luminescence is read using a plate reader. Data is analyzed in Excel and graphed in GraphPad Prism. Statistical analysis and p value is calculated using Mann-Whitney nonparameteric t tests using GraphPad Prism.
• Example 1 demonstrated robust efficacy in a variety of tumor models, with significant regressions noted in the tumor models harboring mutations m ARIDIA, KMT2C or RBI genes. Taken together, these findings show that inactivating mutations in the ARID 1 A, KMT2C, KMT2D or RBI gene presents a potential patient selection strategy for treatment with Example 1 across multiple cancer types.
• F. EMEM CellGro cat#17-305-CV + L-Glutamine + Na Pymvate + Na Bicarbonate + NEAA+ 10% FBS (Gibco cat#10082)
• DD. 1 1 MEM (11095): F12 (CellGro 10-080-CV) + 10% FBS (Gibco cat#10082) EE. RPMI-1640 with glutamine and HEPES (Hyclone Cat # SH30255) + 10% FBS (Gibco Cat # 16000) + IX NaPyr
• HH. 1 1 Hams F12: DMEM + L-Glutamine + 5% FBS (Gibco cat#10082)
• JJ. 1 1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/L sodium bicarbonate and Medium 199 containing a final concentration of 2.2 g/L sodium bicarbonate + 15% FBS
• Table 5 Distribution (%) of anti-proliferation effects of Example 1 across cancer cell lines carrying LOF mutations.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Epidemiology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Nitrogen Condensed Heterocyclic Rings (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Priority Applications (27)

Applications Claiming Priority (6)

Publications (1)

Family

ID=64477309

Family Applications (1)

Country Status (31)

Cited By (2)

Families Citing this family (14)

Citations (5)

Family Cites Families (3)

• 2018
  • 2018-10-31 TW TW107138486A patent/TWI703149B/zh active
  • 2018-11-09 CN CN201880071902.0A patent/CN111344292B/zh active Active
  • 2018-11-09 JO JOP/2020/0122A patent/JOP20200122B1/ar active
  • 2018-11-09 HU HUE18808599A patent/HUE060078T2/hu unknown
  • 2018-11-09 CR CR20200184A patent/CR20200184A/es unknown
  • 2018-11-09 RS RS20220824A patent/RS63530B1/sr unknown
  • 2018-11-09 AU AU2018369508A patent/AU2018369508B2/en active Active
  • 2018-11-09 MD MDE20200963T patent/MD3710446T2/ro unknown
  • 2018-11-09 DK DK18808599.7T patent/DK3710446T3/da active
  • 2018-11-09 EP EP18808599.7A patent/EP3710446B1/en active Active
  • 2018-11-09 WO PCT/US2018/060025 patent/WO2019099298A1/en unknown
  • 2018-11-09 PL PL18808599.7T patent/PL3710446T3/pl unknown
  • 2018-11-09 JP JP2019527416A patent/JP6633254B2/ja active Active
  • 2018-11-09 US US16/185,801 patent/US10472370B2/en active Active
  • 2018-11-09 MY MYPI2020002310A patent/MY197700A/en unknown
  • 2018-11-09 LT LTEPPCT/US2018/060025T patent/LT3710446T/lt unknown
  • 2018-11-09 CA CA3080910A patent/CA3080910C/en active Active
  • 2018-11-09 SG SG11202003677UA patent/SG11202003677UA/en unknown
  • 2018-11-09 KR KR1020207013683A patent/KR102450727B1/ko active IP Right Grant
  • 2018-11-09 MX MX2020004932A patent/MX2020004932A/es unknown
  • 2018-11-09 NZ NZ763551A patent/NZ763551A/en unknown
  • 2018-11-09 BR BR112020008253-8A patent/BR112020008253A2/pt unknown
  • 2018-11-09 PE PE2020001000A patent/PE20210392A1/es unknown
  • 2018-11-09 HR HRP20221152TT patent/HRP20221152T1/hr unknown
  • 2018-11-09 ES ES18808599T patent/ES2925219T3/es active Active
  • 2018-11-09 SI SI201830727T patent/SI3710446T1/sl unknown
• 2019
  • 2019-09-26 US US16/583,661 patent/US10787460B2/en active Active
• 2020
  • 2020-05-07 CL CL2020001218A patent/CL2020001218A1/es unknown
  • 2020-05-08 IL IL274540A patent/IL274540B/en unknown
  • 2020-05-15 EC ECSENADI202025899A patent/ECSP20025899A/es unknown
  • 2020-05-16 SA SA520411990A patent/SA520411990B1/ar unknown
  • 2020-06-01 DO DO2020000102A patent/DOP2020000102A/es unknown
  • 2020-09-22 US US17/028,334 patent/US20210032257A1/en not_active Abandoned

Patent Citations (5)

Non-Patent Citations (8)

Also Published As

Similar Documents

Legal Events